Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection

Wang, Qing S.; Jan, Eric

Each IGR IRES with flanking upstream and downstream sequences was cloned between the EcoRI site and the NcoI site within the intergenic region of plasmid pEJ551 [31] , which was based on the bicistronic construct pRDDEF first described by the Sarnow group [32] . The Firefly luciferarse (FLuc) gene was fused in frame to either the 0 or +1 frame. The sequences that were cloned are as follows: nucleotides 5974-6372 of Cricket paralysis virus (CrPV, accession: NC_003924.1), nucleotides 6372-6908 of Israel acute paralysis virus (IAPV, accession: NC_009025), nucleotides 6296-6814 of acute bee paralysis (ABPV, accession: NC_002548), nucleotides 6381-6908 of Kashmir bee virus (KBV, accession: NC_004807), and nucleotides 4189-4797 of Solenopsis invicta virus (SINV-1, accession: NC_006559). For all IAPV IRES-containing bicistronic constructs, the AUG start codon of the FLuc gene was removed by PCR-based site-directed mutagenesis (Stratagene). In the case of bee paralysis viruses including IAPV, ABPV and KBV, reporter constructs were generated such that the UAA stop codon within SLVI serves as the termination codon for the reporter gene Renilla luciferase (RLuc).